The purpose of this project is to isolate the genes encoding the non-muscle myosins of Acanthamoeba and to use the genes as tools to investigate myosin structure/function relationships and the in vivo functions of these cytoplasmic myosins. This project is part of the general effort in the Laboratory of Cell Biology to understand the organization and function of the cytoskeleton, using as a model system the soil amoeba Acanthamoeba. Acanthamoeba expresses simultaneously at least three distinct myosin enzymes, myosin IA, myosin IB and myosin II. Using molecular cloning techniques, we have isolated and purified a myosin II heavy chain gene and a myosin IB heavy chain gene. This study will provide, for the first time, the complete amino acid sequence of a non-muscle myosin. While non-muscle and muscle myosins share many common features, non-muscle myosins do possess unique structural, enzymatic, and regulatory properties. The amoeba myosin sequence data will be of great value in furthering our understanding of the unique structural and functional aspects of the amoeba myosins, and hopefully provide insight into the properties of cytoplasmic myosins in general. The significance of this work is that by using the tools of molecular biology we can approach the study of these myosins in novel ways which are not possible using the classical techniques of protein chemistry. For example, we can use the genes to (1) make single determinant antibodies to synthetic peptides as probes of myosin function, (2) assign function sites in the 1 degree sequence in combination with the amino acid composition of chemically crosslinked peptides, (3) search for cytoplasmic myosin genes in higher eukaryotes, (4) alter the intracellular levels of myosin in the living cell as a way to study their roles in cell physiology, and (5) study structure/function relationships via site-directed mutagenesis of the gene.